Abstract
The cAMP response element-binding protein (CREB) mediates both basal and PKA-inducible transcription through two separate and independently active domains, the constitutive activation domain (CAD) and the kinase-inducible domain, respectively. The CREB CAD interacts with the general transcription factor TFIID through one or more of the TATA-binding protein-associated factors (TAFs), one of which is TAF110. The CAD is composed of three subdomains, rich in either serine, hydrophobic amino acids, or glutamine. In the present study, analysis of deletion mutants of the CAD showed that all three CAD subdomains were required for effective interaction with TAF110 in a yeast two-hybrid assay. Therefore, a library of random point mutations within the CAD was analyzed in a reverse two-hybrid screen to identify amino acids that are essential for interaction with the TAF. Interaction defects resulted solely from mutations of hydrophobic amino acid residues within the hydrophobic cluster to charged amino acid residues. Together, the deletion and mutation analyses suggest that the entire CAD provides an environment for a specific hydrophobic interaction with TAF110 that is crucial for interaction. Our results provide further evidence for a model of basal activation by CREB involving interaction with TAF110 that promotes recruitment or stabilization of TFIID binding to the promoter, which facilitates pre-initiation complex assembly.
Highlights
The cAMP response element is an enhancer that mediates both basal and cAMP-induced transcriptional activation of a variety of genes in many cell types [1,2,3,4,5,6]. cAMP response element-binding protein (CREB)1 binds constitutively as a dimer to CREs in the promoter of the target gene and activates basal transcription even in the absence of hormonal stimuli [3]
We showed that the CREB constitutive activation domain (CAD) binds TFIID through interaction with one or more TATA-binding protein-associated factors (TAFs), rather than directly with the TATA-binding protein (TBP) [15]
CREB contains two independent activation domains, kinaseinducible domain (KID) and CAD, that were defined by functional analysis of CREB-Gal4 (CRG) fusion proteins (Fig. 1A)
Summary
The cAMP response element is an enhancer that mediates both basal and cAMP-induced transcriptional activation of a variety of genes in many cell types [1,2,3,4,5,6]. cAMP response element-binding protein (CREB)1 binds constitutively as a dimer to CREs in the promoter of the target gene and activates basal transcription even in the absence of hormonal stimuli [3]. When the CREB CAD is fused to the DNA-binding domain (DBD) of the yeast Gal4 protein, it activates basal transcription from a Gal4 promoter nearly as well as the complete unphosphorylated CREB [3, 11].
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